Disruption of endocardial Jagged1-Notch1 signaling blocks EMT and formation of endocardial cushions.

<p><b>A</b>–<b>D</b>, wholemount views showing that at E10.5 the gross morphology was comparable between <i>N1^f/f</i> (<b>A</b>) and<i>N1^f/f;c1^Cre</i> (<b>B</b>) embryos. Similar results were observed between <i>J1^f/f</i> (<b>C</b>) and <i>J1^f/f;c1^Cre</i> (<b>D</b>) embryos. <b>E–L,</b> H&E stained sections through the atrioventricular canal (avc) region of E10.5 embryos showing dense mesenchymal cells (arrows) in the cushion of <i>N1^f/f</i> (<b>E</b> and <b>I</b>) or <i>J1^f/f</i> (<b>G</b> and <b>K</b>) hearts, but fewer mesenchymal cells in the same region of <i>N1^f/f;c1^Cre</i> (<b>F</b> and <b>J</b>) or <i>J1^f/f;c1^Cre</i> (<b>H</b> and <b>L</b>) hearts. <b>M</b> and <b>N,</b> quantitative analysis of the number of mesenchymal cells in the cushion of <i>N1^f/f;c1^Cre</i> (<b>M</b>) or <i>J1^f/f;c1^Cre</i> (<b>N</b>) hearts. *<i>p</i><0.001. <b>O–Q, </b><i>in vitro</i> collagen gel EMT assay showing that while ∼80 endocardial cells (arrows) migrated away from each <i>N1^f/f</i> explant (<b>O</b> and <b>Q</b>), fewer (25/explant) cells underwent this process in cultured <i>N1^f/f;c1^Cre</i> explants (<b>P</b> and <b>Q</b>). *<i>p</i><0.001.</p

Loss of Notch1 in the endocardium is embryonic lethal.

<p><b>A</b> and <b>B,</b> wholemount X-gal staining of the E10.5 <i>R26^fslz;c1^Cre</i> embryo and yolk sac showing that the Cre recombinase-mediated LacZ expression (blue) was restricted to the heart (<b>A</b>) and not present in the yolk sac (<b>B</b>). <b>C,</b> sections of the X-gal stained embryos showing that LacZ expression was localized in the endocardium (arrowheads) and endocardial-derived cushion mesenchymal cells at the atrioventricular canal (AVC). <b>D–G,</b> Immunofluorescence showing that Notch1 protein is present in the AVC endocardium (<b>D</b>, arrowheads) and the pharyngeal vascular endothelium of <i>N1</i><b><i>^f/f</i></b> embryo (<b>F</b>, arrows), but not in the AVC endocardium of <i>N1</i><b><i>^f/f</i></b><i>;c1</i><b><i>^Cre</i></b> embryo (<b>E</b>, arrowheads). Note that Notch1 protein remains in the pharyngeal vascular endothelium of <i>N1</i><b><i>^f/f</i></b><i>;c1</i><b><i>^Cre</i></b> embryo (<b>G</b>, arrows). <b>H</b> and <b>I,</b> Wholemount views showing that E11.5 <i>N1</i><b><i>^f/f</i></b><i>;c1</i><b><i>^Cre</i></b> embryos were runted and had dilated pericardial sac (<b>H</b>) and E12.5, <i>N1</i><b><i>^f/f</i></b><i>;c1</i><b><i>^Cre</i></b> embryos were absorbed (<b>I</b>). <b>J,</b> summarizing the total number of embryos analyzed at different stages, indicating that <i>N1</i><b><i>^f/f</i></b><i>;c1</i><b><i>^Cre</i></b> embryos died between E11.5 and E12.5. The expected number of embryos at different stages is indicated in the parentheses.</p

<i>Bmp2</i> expression is regulated by Wnt signaling.

<p><b>A</b>, qPCR analysis showing that <i>Bmp2</i> expression was inhibited by Wif1 and induced by Wnt4. E9.5 embryos were cultured in the control media or media with Wnt inhibitor Wif1 or recombinant mouse Wnt4. After 24-hour culture, each RNA sample was prepared from AVC tissues of 5 hearts for each treatment. The data from three independent samples for each group were used for statistical calculation. *<i>p</i><0.05 and **<i>p</i><0.01. <b>B–D,</b> RNA <i>in situ</i> hybridization analysis showing <i>Bmp2</i> expression (indicated by arrows) in cultured wild type embryos under indicated condition; cushion myocardial <i>Bmp2</i> expression was inhibited by Wif1 (<b>C</b>) and induced by Wnt4 (<b>D</b>). <b>E–G,</b> RNA <i>in situ</i> hybridization showing <i>Bmp2</i> expression in cultured <i>N1^f/f</i> (<b>E</b>), <i>N1^f/f;c1^Cre</i> embryos (<b>F</b>), and <i>N1^f/f;c1^Cre</i> embryo treated with Wnt4 (<b>G</b>). The data indicated that <i>Bmp2</i> expression was reduced in the <i>N1^f/f;c1^Cre</i> embryo when compared to the <i>N1^f/f</i> embryo. However, Wnt4 treatment restored its expression in the <i>N1^f/f;c1^Cre</i> embryo (<b>G</b>). a, atrium and v, ventricle.</p

Endocardial Jagged1-Notch1 signaling regulates expression of endocardial <i>Wnt4</i> and myocardial <i>Bmp2</i>.

<p><b>A</b>, qPCR analysis of EMT gene expression in the atrioventricular canal (avc) from E10.5 <i>N1^f/f</i> or <i>N1^f/f;c1^Cre</i> hearts. Each cDNA sample was prepared from five avc tissues and three samples of each group were used for qPCR. Gene expression was normalized to <i>Gapdh</i>. *<i>p</i><0.05; **<i>p</i><0.01. <b>B–E,</b> RNA <i>in situ</i> hybridization showing endocardial <i>Wnt4</i> (<b>B</b>, ec, arrowheads) and myocardial <i>Bmp2</i> expression (<b>D</b>, myo, arrows) in E10.5 <i>N1^f/f</i> hearts. Their expression is dramatically reduced in <i>N1^f/f;c1^Cre</i> hearts (<b>C</b> and <b>E</b>). <b>F,</b> qPCR analysis of EMT gene expression in the AVC cushions of E10.5 <i>J1^f/f</i> or <i>J1^f/f;c1^Cre</i> hearts. <b>G–J,</b> RNA <i>in situ</i> hybridization showing that <i>Wnt4</i> and <i>Bmp2</i> expression is downregulated in <i>J1^f/f;c1^Cre</i> hearts. a, atrium and v, ventricle.</p

Wnt4 and Bmp2 act downstream of Notch to regulate EMT.

<p><b>A</b>, showing <i>in vitro</i> collagen gel EMT assay analysis of AVC explants from E9.5 <i>R26^fsGFP;c1^Cre</i> embryos in which the endocardial cells were labeled by GFP (indicated by arrowheads). <b>B</b>, showing that Notch inhibitor DAPT blocked EMT by endocardial cells. <b>C–E</b>, Bmp2, Wnt2, or Wnt4 treatment rescued EMT defect caused by DAPT. <b>F</b>, showing that Bmp inhibitor Noggin abolished the Wnt4 rescuing. <b>G,</b> showing quantitative analysis of the number of transformed mesenchymal cells under each condition. The number of explants analyzed is indicated in parentheses.</p

Working model shows Notch-Wnt-Bmp signaling axis that regulates EMT and early valve formation.

<p><b>A</b>, Schematic showing the cardiac phenotypes found in the endocardial <i>Jagged1</i> or <i>Notch1</i> knockout embryos. During E9.5-E10.5, cushion endocardial cells undergo EMT and form endocardial cushions at the atrioventricular canal and outflow tract of the wild-type (WT) embryos. This process is disrupted in the endocardial <i>Jagged1</i> (<i>J1^f/f;c1^Cre</i>) or <i>Notch1</i> (<i>N1^f/f;c1^Cre</i>) knockout (KO) embryos, which results in hypocellular endocardial cushions. <b>B,</b> Endocardial Jagged1-mediated Notch1 activation induces expression of Wnt4, which subsequently upregulates expression of Bmp2 in the adjacent myocardium. Myocardial Bmp2 then acts on endocardial cells to promote EMT. This Notch-Wnt-Bmp signaling axis promotes EMT during heart valve development.</p

Endocardial-specific deletion of <i>Notch1</i> does not affect early vascular formation.

<p><b>A</b>–<b>C,</b> wholemount view showing the yolk sac vessels in E9.5 <i>N1^f/f</i> (<b>A</b>) and <i>N1^f/f;c1^Cre</i> (<b>B</b>) embryos; they are not present in the <i>N1^f/f;Tie1^Cre</i> embryos (<b>C</b>). <b>D–I,</b> Pecam1 staining showing mature vessels in the yolk sac and head of <i>N1^f/f</i> (<b>D</b> and <b>G</b>) and <i>N1^f/f;c1^Cre</i> embryos (<b>E</b> and <b>H</b>); they are not formed in the <i>N1^f/f;Tie1^Cre</i> embryos (<b>F</b> and <b>I</b>). <b>J–L,</b> wholemount view showing mature yolk sac vessels in E10.5 <i>N1^f/f</i> (<b>J</b>) and <i>N1^f/f;c1^Cre</i> (<b>K</b>), but not in <i>N1^f/f;Tie1^Cre</i> embryos (<b>L</b>). <b>M–R,</b> Pecam1 staining showing mature vascular networks in the yolk sac and head of E10.5 <i>N1^f/f</i> (<b>M</b> and <b>P</b>) and <i>N1^f/f;c1^Cre</i> (<b>N</b> and <b>Q</b>) but not <i>N1^f/f;Tie1^Cre</i> (<b>O</b> and <b>R</b>) embryos.</p

Down-regulation of Skp2 expression inhibits invasion and lung metastasis in osteosarcoma

Abstract Osteosarcoma (OS), the most common primary cancer of bone, exhibits a high propensity for local invasion and distant metastasis. This study sought to elucidate the role of S phase kinase-associated protein (Skp2) in osteosarcoma invasion and metastasis and to explore flavokawain A (FKA), a natural chalcone from kava extract, as a potential Skp2 targeting agent for preventing osteosarcoma progression. Skp2 was found to be overexpressed in multiple osteosarcoma cell lines, including 5 standard and 8 primary patient-derived cell lines. Patients whose tumors expressed high levels of Skp2 sustained a significantly worse metastasis-free (p = 0.0095) and overall survival (p = 0.0013) than those with low Skp2. Skp2 knockdown markedly reduced in vitro cellular invasion and in vivo lung metastasis in an orthotopic mouse model of osteosarcoma. Similar to Skp2 knockdown, treatment with FKA also reduced Skp2 expression in osteosarcoma cell lines and blocked the invasion of osteosarcoma cells in vitro and lung metastasis in vivo. Together, our findings suggest that Skp2 is a promising therapeutic target in osteosarcoma, and that FKA may be an effective Skp2-targeted therapy to reduce osteosarcoma metastasis